G protein-coupled receptor kinase 2 (GRK2) regulates heterotrimeric G protein signaling in the heart not only by phosphorylating activated beta-adrenergic receptors, thereby initiating their desensitization, but also by sequestering activated G protein alpha and betagamma subunits. Despite its beneficial role in adaptation, unusually high expression of GRK2 is strongly implicated in the onset of cardiovascular disease. We recently determined the crystal structure of a peripheral membrane complex between GRK2 and Gbetagamma. The structure reveals the core architecture shared by all GRKs, and is the first description of Gbetagamma bound to a bona fide effector target. This proposal seeks to address some of the questions generated by the GRK2:Gbetagamma structure, particularly those pertaining to the mechanism of activation of GRK2 by Gbetagamma, phospholipids and GPCRs. The first aim is to characterize the ligand binding sites of GRK2 by determining co-crystal structures of GRK2:Gbetagamma in complex with phospholipids or phospholipid head groups, and by determining novel structures of GRK2:Gbetagamma in complex with nucleotide analogs and peptide substrates. We will also model peptides corresponding to known GRK2 phosphorylation by docking them to the kinase domain, not only to learn more about the sequence specificity of GRK2, but also potentially to develop better peptide substrates or inhibitors. The second aim is to define the conformational changes induced in GRK2 by the binding of Gbetagamma, primarily by determining the structure of the cytosolic form of GRK2 from existing crystals. In addition, conformational changes in GRK2 induced by ligands or membrane translocation will be evaluated using limited proteolysis and ligand-binding assays. The third aim is to define the receptor-docking site of GRK2. First, site-directed mutants of various residues within the predicted docking site will be tested for their ability to block receptor phosphorylation. Secondly, methods to improve existing crystals of the complex between GRK2:Gbetagamma and the activated beta2-adrenergic receptor will be developed, with the ultimate goal of determining its crystallographic structure. In an alternative approach, structures will be determined of GRK2:Gbetagamma in complex either with peptides that correspond to one or more cytosolic loops of the beta2-adrenergic receptor or with a peptide, mastoparan, that mimics the catalytic activity of GPCRs.